World Applied Sciences Jownal9 (3): 268-274,2010

ISSN 1818-4952
mOSI Publications, 2010
Assessment of Inhalation Exposure to Amitraz among
Pesticide Sprayers in Zangiabad, Iran
1,2MajidAghasil, lZailina Hashim, 3MitraMehrabani, 4Dzolkhijli Omar and lSaidiMoin
lFaculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM), Malaysia
'Faculty of Health, Kennan Medical University (KMU), Iran
3Herbal and Traditional Medicine Research Centre, Kerman Medical University (KMU), Iran
4Faculty of Agriculture, Universiti Putra Malaysia (UPM), Malaysia
Abstract: Orchard operations involve heavy use of pesticides to control pests and the potential for exposure
of sprayers is high. "When high pressure equipments for pesticide application are used, the potential for
respiratory exposure is increased. The aim of this study was to assess the amitraz residue level in the individual
breathing zone of the sprayers. Individual air samples of 70 amitraz sprayers have been collected using modified
fitted with irnpinger acetonitrile as liquid sorbent. A sample size of 480 liters of air was collected in each case.
The mean concentration of amitraz and its metabolite in the breathing area during the application were 11.51 and
135 fig/m', respectively,
Key words: Occupationally Exposure . Arnitraz . Orchard Operation' Pistachio' Iran
INTRODUCTION
Amitraz (N-(2, 4-dimethy Ipheny l)-N -[ (2, 4-
dim ethy 1 pheny 1) - im ino 1 m ethy I-N-m ethy 1 in
ethanimidamide) is a member of formamidine class
chemical family [1, 2]. It is a non-systemic acaricide and
insecticide, whereby the contact and respiratory actions
is used to control insects, ticks and mites [3, 4]. Through
a series of intermediate compOlmds, amitraz hydrolyses to
form an environmentally stable compOlmd called 2,4-
dirnethylaniline (2,4-DMA) [3], The degradation of amitraz
to stabilize aniline products may significantly contribute
to the environmental and health risks involved in the
application and use of this particular pesticide [5]. More
importantly, 2,4-dimethylaniline is also toxic, with an acute
oral LD50 of 467 mglkg for rats, which is ahilost half that
of the parent pesticide [6],
Iran is the largest producer of pistachio in the world,
accOlmting for about two-thirds of the global planted
areas and slightly more than one-half of the world's
production in the recent years [7]. About 13 different
pests and diseases have been fOlUld to attack pistachio
and 1,800 tonnes of pesticides are used for this particular
crop annually [8]. Like many other developing cOlUltries,
Iran has promoted the use of pesticides to expand
agricultural land and increase output per acre. Over 27,000
tonnes of pesticides were used in the Islamic Republic of
Iran in 2000101 and the country spent US$125 million on
pesticide imports in 2002 [8],
The potential risk of exposure to pesticide residues in
working environments is high [9]. For airborne
contamination measurements, close attention must be
paid to the breathing zone [10], Pesticides may be inbaled
in efficient dose to cause serious damage to respiratory
tract or to be absorbed through the llUlgs and into the
bloodstream. It is crucial to note that the hazard of
poisoning from respiratory exposure is great because of
the rapid and complete absorption of pesticides through
llUlg tissues. LWlgs may be exposed to pesticides by
inhalation of powders, airborne droplets or vapours. The
hazard from inhalation of pesticide spray droplets is fairly
low when diluted sprays are applied with conventional
low pressure application equipment. However, when high
pressure equipments for pesticide application are used,
the potential for respiratory exposure is increased. Some
non-fumigant pesticides are toxic to pests as liquid or
solid formulations, but they also give off vapours which
could be toxic to applicators [Ill
Corresponding Author: Zailina Hashim, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM), Malaysia.
Tel: +603-89472406 & 6017-6361367; E-mail: zailina@medic,upmedu,my
268
WorldAppl. Sci. J, 9 (3): 268-274, 2010
In addition, orchard operations involve heavy use
of pesticides in controlling pests and the potential for
workers to be exposed to them is high. Safety measures
for applicators are still very poor. Many of them do not
strictly follow the manufacturer's directions in using the
formulations [9]. Respiratory exposure cannot be
completely separated from oral or dermal exposure in the
sense that some materials which are retained on the
mucous membranes of the upper respiratory system will
be absorbed through these membranes or swallowed and
made available for absorption by the gastrointestinal tract.
However, the error is on the side of greater safety if all
inhaled material is assumed to represent respiratory
exposure, since most, if not all, materials are absorbed
more rapidly and more completely through the hmgs than
through the skin and, for this and perhaps other reasons,
are more toxic by the respiratory route [12].
Amitraz replaced zolon for pistachio pest control in
Iran about 15 years ago and since then, this particular
pesticide has been used in Iran, with approximaly several
tonnes of use per year. During that period, amitraz was
not regulated and agronomists and farmers had little
knowledge about the hazardous risk of using this
particular pesticide. In Iran amitraz is available Wlder the
proprietry name Mitac with 20% emulsifable amitraz
concentrate. Although information on the presence of
pesticides in the atmosphere is available [13, 14], data
related to determination of pesticides in orchards is still
limited, while reports on the assessment of amitraz in
pistachio orchards is not fOlUld in the literature. The aim
of this study was to assess the amitraz residue and
metabdite level in the individual breathing zone of the
sprayers.
METHODOLOGY
Study Area: Zangiabad is a small city, located 20 km to
the north of Kerman, which is the capital of Kerman
province. This city is located in an arid desert area with an
average arumal rainfall of 135mm. The locale is also well-
kno"\iVIl for growing pistachio nuts. The city is located on
a flat plain with an area of 10 lan
2
with 5280 hectares of
pistachio farms. Arnitraz is the most frequently used
pesticide in the Zangiabad area for pistachio pest control.
The exposure to amitraz may be of occupational origin or
strongly related to environmental contamination.
Study Location: This cross-sectional study was carried
out in the pistachio orchards in Zangiabad area. The data
collection was carried out from April to September 2008
269
according to the expected frequency and duration of
amitraz spraying in the study area. Mature pistachio trees
are planted in 6.00-m rows with 3.00-m tree spacing.
Individual trees have a height of about 2.50-m and 1.50 m
width. The applicators used tractor with high pressure
application equipment and a nozzle operating at a flow
rate about 15 L per minute for spraying of amitraz. The
volume sprayed was 4000 L in each case, corresponding
approximately to a dose of 1.5 L per hectar amitraz. The
spray liquid was prepared by dispersing 4 L of amitraz
20 % EC in a tank containing 4000 L of water.
Air Sampling: Individual samples of amitraz were
collected using liquid sorbent and modified fritted
impinger in order to determine the air amitraz
concentration. The SKC impinger with a fritted nozzle
was modified. The head and stem of impinger was
adapted to a 250 mL round bottom flask. The modified
impinger, with a mini pump air sampler was used for air
sampling. Based on Briand et al. [15], the airflow was 2
litres/minute of air sampling. The impinger was filled
with 60 mL acetonitrile. Acetonitrile was handled
carefully, because it can be a hazard ous when the
impinger is mOlUlted on a worker. In order to protect
the pump from splashed or spilled impinger liquid, a
standard impinger which served as a trap was installed
between the impinger and the pump. The sampling device
containing the modified impinger, trap and mini pump air
sampler was portable. This device was fitted to each
sprayer (in front of the chest) with two belts. One belt was
put aroWld the neck while the other was securely placed
behind the chest. For the comfort and safety of the
participants, the sampling equipment was attached to the
sprayer so that it would not interfere with their
performance or safety.
After sampling, the stopper on the impingers was
tightly sealed with parafihn to prevent leakage while
travailing to the laboratory. As soon as they were
returned to the laboratory from the field, all the samples
held in a cold box were stored in the dark at 4 0c. With
each batch of ten samples, one midget impinger
containing the same volume of acetonitrile, prepared from
the same stock as that used for the sample collection, was
submitted as the blank. This impinger was subjected to
exactly the same handling as the samples except that no
air was dra"\iVIl.
The air samples were collected only during the active
spraying phase of each simulation. The pumps were
calibrated in the laboratory before going for field
operation. A population group of 70 amitraz sprayers
WorldAppl. Sci. J, 9 (3): 268-274, 2010
was studied. The origin of the exposure was the use of
amitraz on pistachio trees. The sprayers was not permitted
to participate in any activity other than spraying. The inlet
of impingers was kept in the breathing zone of the
applicator and was operated for a period of four hours. A
sample size of 480 htres of air was collected. The air was
sampled at a flow rate of 2 litres per minute for four hours.
In order to prevent the breakthrough of amitraz, the
impingers were changed after 2 hours during the field test.
The flow rate of the personal sampler pump was checked
at the beginning and the end of the exposure period with
an SKC calibrator. Pesticide sprayers did not handle
concentrated amitraz, but they only handled diluted
pesticide which was ready to spray. After air sampling,
some protective equipment such as mask and gloves were
given to each pesticide operator as an educational health
activity to encourage them to use the protective
equipment.
Air Samples Extraction Procedure: After sampling, a
solvent concentration step was made. At first, the
stem of the impingers was rinsed with 2 mL of
acetonitrile in the midget impinger flask in the laboratory
and this was repeated. The rOWld-bottom flask was
attached to a rotary evaporator and the sample was
evaporated to aroWld 3.0 mL at 50e. The sample
solution for each impinger was transferred into a separate
6.0-mL glass tube with a Teflon cap and 1.5 mL of
acetonitrile was used to wash each impinger. This process
was repeated and combined with an appropriate sample
solution. After that, the solvent was removed Wlder a soft
stream of nitrogen gas for five minutes without heating.
The evaporation process was stopped when 1.0 ml of
solution was left. A 20-).11 volume of internal standard
solution thymol (500 ng/ mL in acetonitrile) was
added into the extract and the cap of glass tubes
were then kept tight and wrapped with an aluminium
foil and these were shipped out for analysis immediately.
Finally, quantification and confirmation of the results
were made USIng a gas chromatography-mass
spectrometer (GC-MS).
GC-MS Analysis: The concentrated stock solution of
1 ).1g per mL was prepared by diluting pure amitraz and
2,4-dimethylaniline in acetonitrile. The extract was
analyzed using the GC-MS. A 1 microliter aliquot of the
sample solution was injected into the gas chromatograph.
The syringe was cleaned with pure acetonitrile and dried
thoroughly between injections. The syringe was then
ready to take up the sample for injection. The injection
270
was repeated for each sample. The peak area was
measured by the area Wlder the resulting peak and
compared with the areas obtained from the injection of
standards to prepare calibration curve, as discussed
below.
GC-MS Apparatus and Conditions: The analysis was
carried out on a GC system coupled with quadrupole mass
spectrometer (GCMS-QP5050, Shirnadzu Corporation,
Japan). The compoWlds were separated on the ZB-
Multiresidue-1 capillary column (Phenomenex, USA,
3Omx0.25mm i.d. x 0.25flm fihn thickness). The injection
temperature, GC-MS interface and IOn source
temperatures were 280, 230 and 230C, respectively.
Meanwhile, the GC oven temperature program utilized an
initial temperature of 1 00 C and an initial holding time of
5 min and the temperature was increased from 20C/min
to 136C, at which it was held for 2 min and was then
increased from 20C/min to 300C and held for 5 min.
Relum was used as carrier gas with a linear speed of
25 cm/s. Amitraz and its metabolite 2,4-dimethylaniline
were analyzed in selected full scan mode. The ionizing
energy was 70 e V. A 1 ).lL aliquot of each extract was
injected into gas chromatograph. The injection was
splitless and the mass spectrometer was calibrated
weekly.
Calibration Curves: An eight-point standard calibration
curve was made using the analysis of amitraz and 2,4-
dimethylaniline. Standard solutions of both analytes were
prepared by dissolving the above compoWlds in
acetonitrile to yield the final concentrations of 50, 250,
500, 1000, 2000, 4000, 8000 and 10000 ng/mL.
Thymol (500 ng/mL) was used as internal analytical
standard. Addition of only acetonitrile ( C ~ O ) was
used as control. Meanwhile, the peak area ratio (PAR)
was obtained from the GC-MS analysis of each
compoWld at different concentrations (ng/mL). After that,
the calibration curves were constructed by plotting with
the peak area ratio of the analytes and IS on the Y-axis
and the concentration on the X-axis.
Calculation: The analyte concentrations for samples were
obtained from the calibration curve in terms of micrograms
of amitraz per sample. The air concentrations were
calculated using the following formulae:
(micrograms ofamitraz per sample) * (1000)
Jlghn3 = ------------------------------------------------------------
(liters of air sampled)
WorldAppl. Sci. J, 9 (3): 268-274, 2010
Recovery Efficiency: The extraction recovery was
determined by comparing the peak area ratios of amitraz
and 2,4-dirnethylanihne with the IS of the extracted
samples with the peak-area ratios obtained from a direct
injection of a standard solution containing the same
concentration of amitraz, or its metabolite and the IS
(500 ng). In order to determine recovery, three irnpingers
were spiked with analyts to yield 0.05,0.5 and 5.0 fig/mL
concentrations. Amitraz and 2,4-dirnethylaniline were
diluted in acetonitrile and then extracted according to the
same procedure as previously described. Seven replicates
were made at each fortification to calculate the mean and
standard deviation for recovery. At the same time, a
parallel blank was also prepared except that no sample
was added to it. The recovery efficiency was calculated
using the following equation [16]:
(oe /IS )
Recovery% = ewact ewad X 100%
(OC'Pil!JJ I IS,piI!JJ)
Where;
OC extract = The peak area for the organic compOlmd
(OC) in the extract,
IS extract = The peak area for the internal analytical
standard in the same extract,
DC spike = The peak area for the OC in the spike
solution and
IS spike = The peak area for the internal analytical
standard in the same spike solution.
RESULTS
The amitraz cover spray was applied by sprayers
who were moving arOlmd the trees, directing spray
into foliage and ensuring it was wetted. A single
medium-sized tree was sprayed in arOlmd 1 min. During
spraying, there was visible overs pray which was carried
by the wind several metres from the point of application.
Splashes of the spray from foliage also contributed to
Table 1: Inhalation Exposure of Amitraz
Parameter
Amitraz (Jlg/m
3
)
Metabolite (Jlg!m
3
)
n
70
70
Minimum
8.89
.89
Table 2: Oral AD!, NOEL and Computed Inhalation Intake values for amitraz
the overspray. With respect to the protective equipments
used during pesticide application, none of the sprayers in
Zangiabad zone used the protective equipments that
normally are required as safety devices. Nobody used
gloves, masks, plastic cover, boots, apron and waterproof
garment.
The average extraction efficiency for seven impingers
spiked at the target concentration was 97.3 % and 97.9 %
for amitraz and its metabolite, respectively. The average
recovery values obtained were at least 95.2 % and as
such, no recovery correction factor was used in the
determination of the true values. Amitraz and its
metabolite were detected in all inhalation air samples of
sprayers. The arithmetic mean (AJ\.1) and standard
deviation (SD) of the concentrations of the target
chemicals in the air samples collected are given in
Table 1. Amitraz and 2,4-dirnethylaniline concentrations
in the air samples are expressed as ).lg analyte per cubic
meter of the air sampled (fig/m').
The mean concentrations of amitraz and its
metabolite in the breathing area during the application
were 11.51 and 1.35 ).lg/m
3
, respectively. The average
retention time was 22.65 min and 7.11 min for amitraz
and 2,4-dimethylaniline, respectively. The following
chromatogram (Figure 1) is from injection oflOOO ng/mL
amitraz and 1000 ng/mL 2,4-dimethylanihne standards
equivalent to 2.08 fig/m' of a 480-L air sample for both
analytes.
Figure 2 illustrates a chromatogram of a breathing air
sample from an amitraz applicator. The concentrations of
amitraz and its metabolite in this particular sample were
10.8 and 1.26 ).lg/m
3
, respectively. The retention time in
this chromatogram was 22.66 min for amitraz and this was
7.11 min for its metabolite.
In order to evaluate the health hazard involved
while working with amitraz, the data of the airborne
concentration of this chemical was compared with the
toxicological limits. Assuming 5 m
3
as the respiration rate
Maximum
14.06
1.82
11.5137
1.3547
StdDeviation
1.33070
.25521
Parameter Om1 AD! (mglkg'd) Oral AD! (mg/d) Oral NOEL (mgikg!d) Oral NOEL (mg!d) Computed Inhalation Intake (mg!d)
Amitraz 0.003 (human) 0.25 (dog)
Adult (70 kg body weight) 0.21 17.5 0.06
(Adaptation: EXTOXNET, 1995; U.S.EPA, 1996)
271
WorldAppl. Sci. 1.,9 (3): 268-274, 2010
int ensin

l50(o()']Oa-

lOOO()I}OIl-
-:"= (o()O 0 0
10
N TIC
.j
:0
lllin
Fig. 1: Chromatogram of Standard Solutions of Amitraz and 2,4-dimethylaniline in an Air Sample
inteni'ity

-'10<:)(")00-
3:"={)OI]OO
30()OI)OO-
:: :"={) (II] 0 0
:0<:)(,,)00-
1 0
10<:)(")00-
0
TIC
:::0
nun
Fig. 2: Typical Chromatogram ofthe Inhalation Air Sample of an Amitraz Applicator, Zangiabad Area, Iran, 2008
for the average adult per four-hours working [17, 18]
and using the concentration of 11.51 Ilg/m3 (Table 1),
the inhalational intake per working day is equivalent to
57.55 Ilg/d or 0.057 mg/d. Hence, with the inhalation
uptake assumed to be 100 % of the applied dose [19],
the daily inhalation exposure for amitraz sprayers would
be 0.057 mg.
272
DISCUSSIONS
The production of pistachio III orchards is
associated with specific pest control problems that result
in numerous applications of plant protection products.
Pistachio trees require intensive care and therefore,
sprayers and agricultural workers are frequently exposed
WorldAppl. Sci. J, 9 (3): 268-274, 2010
to pesticides in the orchard atmosphere. Methods for the
determination of amitraz in air have not been previously
described. A general design criterion for a personal
sampling device is that it should be small and compact so
that the normal daffy flUlCtiOns and jobs can be
accomplished with little or no interference from this
sampling device [20]. Low-volume samplers which are
generally used for personal monitoring are portable,
battery operated, relatively quiet and easy to use. Flow
rates of 0.5-1.5 L/min are typically recommended for
pesticides [21]. In the present study, the applied airflow
was 2 L / min as compared to 1.5 L / min to ensure the
collection of all the atmospheric phases of amitraz.
Different air sampling methods reported for pesticide
determination involve the use of liquid or solid
adsorbents and filters [22]. Most field comparisons found
that impinger and bubbler methods gave higher results
compared to solvent-free methods [23]. Midget impingers
or bubblers collect pesticides as aerosols or vapours, but
are not well suited to personal sampling since they are
cumbersome, breakable and the liquid medium frequently
spills during normal work movements. Filters trap aerosols
but they do not retain pesticide vapours. Solid sorbents
retain pesticide vapours but they may not efficiently
collect or trap aerosol forms [20]. Based on these
limitations, an air collection method using an impinger
which is connected to personal samplers was applied for
assessing potential inhalation exposure to amitraz in the
present study.
To design the air sampler, optimising the
volatilisation was the first step, whereas the shape of the
impinger was important for minimizing the sample loss
caused by volatilization which could occur during
extended sampling periods. Thus, a round flask of 250 mL
was chosen for impinger. In the same way, Durham and
Wolfe [12] stated a method for sampling the air using the
modified impinger and 500-mL Pyrex glass ball. The
second step was to choose a proper solvent to be used in
the impinger. The most suitable medium for a particular
investigation is dependent on the chemicals being
studied. The medium should entrap a high percentage of
the chemical passing through it and allow the elution of a
high percentage of the entrapped chemical for analysis.
The chemical should be recovered without any
conversion to other reaction products and the medium
should not produce a significant restriction of airflow [24].
Since early 1970s, ethylene glycol has been used as a
standard media for collecting pesticides in air [15].
Cyclohexane has also been used in impinger for air
collection [26]. Amitraz is lUlstable in pure methanol but
273
it is stable in acetonitrile [26]. For this reason, acetonitrile
was chosen as a suitable solvent for air sampling of
amitraz. The sampling equipment was attached to the
amitraz applicators so that it would not interfere with their
work performance or safety.
Some studies have shown that both the type of the
collection liquid and the volume affect the collection
efficiency. A higher level of liquid means there is more
time between bubble formation at the fritted tip and
bubble bursting at the surface of the liquid and, thus,
more time for particles to diffuse from the air inside the
bubbles into the liquid [27]. For this reason, impingers
were filled with 60 mL acetonitrile in this study. The
results of a study by Haraguchi e/ al. [14] showed that
many pesticides exist in a gaseous state rather than in a
solid state in air.
In order to evaluate the health hazard involved while
working with toxic compolUlds such as pesticides, it is
important to assess the amolUlt of exposure workers
lUldergo while operating the pesticides. After determining
the concentration of a pesticide in the air, the respiratory
exposure of an exposed person can be calculated using
an assumed tidal volume and respiratory rate [12]. A
comparison with the ADI was also calculated although
the ADI (mglkg body wt/day) refers to oral adsorption.
Table 2 summarizes Acceptable Daily Intake (ADI) and
No Observed Adverse Effect estimates (NOEL) and
computed inhalation intake values for amitraz.
CONCLUSION
The oral No Observed Effect Level (NOEL) in a 70 kg
adult is 17.5 mg/d. Since the mean concentration of amitraz
in pesticide sprayers was 11.51 ).lg/m
3
and the inhalation
intake in this study was 0.06 mg/d, is lower than the
acceptable daily intake for adults of 0.21 mg/d, it appears
that no serious and urgent risk is to be expected and acute
poisoning will not occur due to amitraz exposure.
However, this chemical agent and its metabolites may
cause chronic adverse health effects after a long period of
continuous exposure.
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